Commercial influenza vaccines vary in HA-complex structure and in induction of cross-reactive HA antibodies

Influenza virus infects millions of people annually and can cause global pandemics. Hemagglutinin (HA) is the primary component of commercial influenza vaccines (CIV), and antibody titer to HA is a primary correlate of protection. Continual antigenic variation of HA requires that CIVs are reformulated yearly. Structural organization of HA complexes have not previously been correlated with induction of broadly reactive antibodies, yet CIV formulations vary in how HA is organized. Using electron microscopy to study four current CIVs, we find structures including: individual HAs, starfish structures with up to 12 HA molecules, and novel spiked-nanodisc structures that display over 50 HA molecules along the complex’s perimeter. CIV containing these spiked nanodiscs elicit the highest levels of heterosubtypic cross-reactive antibodies in female mice. Here, we report that HA structural organization can be an important CIV parameter and can be associated with the induction of cross-reactive antibodies to conserved HA epitopes.


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Sample size
Data exclusions Replication Randomization Blinding The source data underlying Figs. 2a,3j,and 4g,5a,c,d,e,f and 6b,c,d,e and Supplementary Figs. 4i,j,5c,and 6a,b,d  Sizes of animal groups were minimized for ethical concerns but still provide statistical significance. Since the effect size of the immune response following vaccination was unknown a power analysis was inappropriate. Since one way ANOVA was going to be the statistical test outcome measure, determining appropriate degrees of freedom was possible and utilizing the resource equation method was a suitable alternative. Based upon the resource equation method, the degree of freedom for the final ANOVA should be a minimum of degrees of freedom/number of groups+1 (10/5+1=3) and a maximum of degrees of freedom/number of groups+1 (20/5+1=5), so 5 mice per group were selected for mouse challenge studies and 4 were selected for pathology. Arifin WN, Zahiruddin WM. Sample size calculation in animal studies using resource equation approach. Malays J Med Sci. 2017;24(5):101-105. https://doi.org/10.21315/mjms2017.24.5.11 1-2 rabbits were vaccinated per vaccine type for sera generation due to limitation based upon the amount of protein necessary for their multiple immunization schedule.
No data were excluded from the analyses.
The biological replicates were stated in each figure legend (n= ). The mouse lethal challenge experiment was replicated twice with earlier endpoints for tissue collection, the bodyweight data for the first 3 days following challenge was consistent between the three experiments. Generation of immunized rabbit sera was conducted once, however prior to termination rabbit sera was tested and confirmed production of antibodies consistent to those from previous mouse immunization experiments.
For all experiments animals (mice or rabbits) were randomly assigned to treatment groups either: vaccine, saline, or uninfected control groups.
An unbiased blinded then unblinded approach was used to analyze data collected from mouse lung tissue: histopathology and TCID50. Mouse daily observations following challenge were conducted blindly, however mouse daily weights were collected by an unblinded experimenter due to pragmatic reasons. Rabbit immunizations experiments were done blind to experimental group. All of the serological assays including ELISA, western, tiering, and structural, biochemical and biophysical characterization of the antibodies were not performed in a blinded manner.
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Anti-NA antibody ThermoFisher Scientific Influenza A H1N1 NA Polyclonal Antibody PA5-32238 was used a 1:1000 dilution. Anti-H1 polyclonal PS6000 (Protein Sciences) was used at 1:1000 dilution. Anti-H3 polyclonal PS6001 (Protein Sciences) was used at 1:1000 dilution. Anti-HB polyclonal PS6003 (Protein Sciences) was used at 1:1000 dilution. Anti-M1 monoclonal HB64 (American Type Culture Collection clone M2-1C6-4R3) was used at 3.5 µg/ml. Anti-NP monoclonal HB65 (American Type Culture Collection clone H16-L10-4R5) was used at 3.5 µg/ml. Antibody C179 (mouse) was first described in PubMed ID 7682624. FI6v3 (human) was first described in PubMed ID 21798894. CR6261 (human) was first described in PubMed ID 19079604. Protein Sciences antibodies were validated for western and ELISA applications. HB64 is described in 78 citrations at ATCC, and HB65 is described in 392 citations at ATCC. No additional validation was conducted prior to experimentation.
The cell lines were not authenticated.
Cell lines were not tested for mycoplasma contamination.
We did not use any commonly misidentified lines.
Female BALB/c mice (Taconic Biosciences) aged 8-10 weeks at experimental start were utilized in these experiments. Mice were maintained in a facility on a 14 hours light and 10 hours dark cycle at an ambient temperature of 22 ± 3°C, and a humidity between 30-70%, recorded daily. New Zealand White rabbits (Cocalico Biologicals) began experimentation at approximately two to four months of age.
The study did not involve wild animals.
Female mice were used for all experiments based upon the higher immunogenetic response following vaccination. Fink AL, Engle K, Ursin RL, Tang WY, Klein SL. Biological sex affects vaccine efficacy and protection against influenza in mice. Proc Natl Acad Sci USA. 2018;115(49):12477-82. Male rabbits were utilized and displayed a similar immune profile to the mouse studies.
The study did not involve samples collected from the field.
All mouse experiments were preformed under protocols approved by the Animal Care and Use Committee (ACUC) at the National Institute of Allergy and Infectious Disease (NIAID). The studies were performed in accordance with all federal regulations, NIH guidelines, AAALAC, and IACUC approval. Rabbit studies were conducted by Life Sciences Solutions (Thermo Fisher Scientific) and were in accordance with the ACUC of Cocalico Biologicals, Inc.